Project Abstract/Summary PRDMs (Positive Regulatory Domain) are chromatin modifiers that epigenetically regulate gene transcription through their histone methyltransferase activity or by forming complexes with other proteins and histone- modifying enzymes. PRDMs are crucial in processes that need precise spatially and temporally controlled gene expression. The formation of the craniofacial skeleton requires proper orchestration of various cellular processes in cranial neural crest cell (cNCC) development, including cell migration, proliferation, differentiation, polarity and cell death. Any alterations that occur during the process of cNCC development are associated with congenital defects and craniofacial abnormalities such as cleft lip with or without cleft palate. These birth defects are very common, often occurring at alarming rates (1:1000 live births annually in the US alone). Therefore, it is necessary to understand the gene-regulatory networks that control cNCC development in order to better understand how disruption of these processes leads to craniofacial defects. Several PRDMs have been linked to craniofacial disorders. Human genome-wide association studies have identified mutations in PRDM3 and PRDM16 linked to patients with cleft palate. Studies in zebrafish and mice have shown loss of Prdm1, Prdm3 and Prdm16 causes hypoplasia of cartilage skeletal elements and changes in the cranial ganglia in addition to cleft palate. The proposed research will examine the importance of Prdm1a, Prdm3 and Prdm16 during formation of the craniofacial skeleton. In two focused aims, this proposed study will define the regulatory roles of Prdm1a, Prdm3, and Prdm16 in cNCC development. Preliminary data in zebrafish suggests loss of prdm1a, prdm3, and prdm16 causes only mild craniofacial defects, in large part due to genetic compensation between all three genes. In addition, initial transcriptional profiling studies led to the central hypothesis that prdm1a, prdm3, and prdm16 genetically compensate for each other and share dual roles in cNCC development: (1) mediating expression of genes associated with maintaining cell polarity and (2) controlling genes that specify cNCC cell fates. In Aim 1, the mechanism(s) responsible for cNCC chondrocyte cellular condensation/polarity and regulation of different cNCC fate decisions with loss of prdm1a, prdm3, and prdm16 will be investigated. In Aim 2, the changes in the chromatin landscape in prdm3- and prdm16-deficient cNCC will be assessed at putative Prdm3 and Prdm16 neuronal and cell polarity target genes as well as other direct target genes identified by Prdm3 and Prdm16 ChIP-seq experiments. This study will provide new insights into the molecular pathways and targets regulated by Prdm1a, Prdm3 and Prdm16 during cNCC development and the formation of the craniofacial skeleton. A better understanding of Prdm1a, Prdm3, and Prdm16 in cNCCs will advance the current knowledge of how gene expression and cellular pathways, such as chondrocyte cell polarity and cNCC fate decisions, are coordinated during craniofacial development. Such information will be essential in better understanding how neural crest associated congenital defects arise.